RESUMO
Bone grafting is a fundamental dental surgery procedure widely used for implant placement and periodontal disease management treatments. Despite its broad applications, vertical bone augmentation presents unique challenges, including the risk of graft displacement due to gravitational and masticatory forces. Traditional physical stabilization methods introduce additional complexities and risks, underscoring the need for innovative fixation technologies. This study aimed to develop an in situ photo-crosslinkable bioadhesive hydrogel (iPBAH) as a multifunctional bone graft binder to enhance the process of bone reconstruction. The bioadhesive is composed of mussel-derived adhesive protein (MAP) fused with the cell-adhesive peptide RGD. The numerous tyrosine residues in MAP facilitate rapid photo-crosslinking, enabling efficient hydrogel formation using visible blue light. Subsequently, iPBAH underwent comprehensive characterization to evaluate its suitability as a multifunctional bone graft binder. iPBAH efficiently underwent in situ crosslinking through harmless exposure to visible light within minutes and displayed several exceptional properties, including a microporous structure, underwater adhesion, extended durability, high compressive strength, and biocompatibility. In vivo assessments, using male Sprague-Dawley rats, demonstrated that iPBAH binder significantly enhanced bone regeneration in a rat calvarial bone defect model. The in situ crosslinking of the iPBAH binder during bone graft transplantation can effectively fill irregular and complex defect shapes while simultaneously preventing graft material leakage. The improved physical attributes of the bound graft material can enhance its resistance to external forces, thereby ensuring sustained retention over time. Moreover, the interaction between iPBAH and surrounding tissues promotes adhesion and integration of the graft material with host tissues in the defect area. In addition, the included RGD peptide in iPBAH can augment inherent cell recruitment, adhesion, and growth, consequently expediting osteogenesis.
Assuntos
Transplante Ósseo , Proteínas , Ratos , Masculino , Animais , Ratos Sprague-Dawley , Osteogênese , Regeneração Óssea , HidrogéisRESUMO
This study was to investigate whether the sexual abstinence period (SAP) recommended by the World Health Organization (WHO) affects clinical outcomes. We compared the rate of clinical outcomes between 2-7 and ≥8 days of SAP in first fresh embryo transfer after intracytoplasmic sperm injection (ICSI) in groups of young maternal age (YMA: <38 years) and old maternal age (OMA: ≥38 years). We conducted a retrospective study of 449 first ICSI cycles with a normal ovarian response. SAP was identified before collecting the semen samples. Semen analysis was performed based on the guidelines recommended by WHO (2010). Sperm preparation was made using the swim-up method. Patients' baseline characteristics in the YMA and OMA groups did not differ. The rates of fertilisation, top-quality embryos on day 3, biochemical pregnancy, clinical pregnancy, ongoing pregnancy, abortion and implantation per cycle were not significantly different between 2-7 and ≥8 days of SAP in the YMA or OMA group. In conclusion, SAP beyond the recommended period by WHO was not associated with the rates of a lower fertilisation and pregnancy in human in vitro fertilisation (IVF). We think that a new criterion of SAP for clinical application in human IVF needs to be considered by WHO.
RESUMO
Members of the Iridoviridae family have been considered as aetiological agents of iridovirus diseases, causing fish mortalities and economic losses all over the world. Virus identification based on candidate gene sequencing is faster, more accurate and more reliable than other traditional phenotype methodologies. Iridoviridae viruses are covered by a protein shell (capsid) encoded by the important candidate gene, major capsid protein (MCP). In this study, we investigated the potential of the MCP gene for use in the diagnosis and identification of infections caused Megalocytivirus of the Iridoviridae family. We selected data of 66 Iridoviridae family isolates (53 strains of Megalocytivirus, eight strains of iridoviruses and five strains of Ranavirus) infecting various species of fish distributed all over the world. A total of 53 strains of Megalocytivirus were used for designing the complete primer sets for identifying the most hypervariable region of the MCP gene. Further, our in silico analysis of 102 sequences of related and unrelated viruses reconfirms that primer sets could identify strains more specifically and offers a useful and fast alternative for routine clinical laboratory testing. Our findings suggest that phenotype observation along with diagnosis using universal primer sets can help detect infection or carriers at an early stage.
Assuntos
Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/diagnóstico , Iridoviridae/genética , Animais , Proteínas do Capsídeo/genética , Infecções por Vírus de DNA/genética , Doenças dos Peixes/virologia , Peixes/virologia , Iridovirus/genética , Filogenia , Ranavirus/genética , Análise de Sequência de DNARESUMO
Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self-renewal and pluripotency, its high cost has limited previous studies, and the development of a low-cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo-derived porcine blastocysts on 5.0 × 105 mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium-based medium for primary culture, on 2.5 × 105 MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5 × 105 MEF feeder cells in DMEM/Ham's F10-based medium for the post-2nd subpassage could support the establishment and maintenance of porcine ES-like cells at the low concentration of bFGF. The established porcine ES-like cells showed ES cell-specific characteristics such as self-renewal and pluripotency. We confirmed that porcine ES-like cells could be generated from in vivo-derived porcine blastocysts at a low concentration of bFGF.
Assuntos
Técnicas de Cocultura/veterinária , Células-Tronco Embrionárias/citologia , Sus scrofa/fisiologia , Animais , Blastocisto/citologia , Técnicas de Cocultura/métodos , Embrião de Mamíferos , Células Alimentadoras , Feminino , Fator 2 de Crescimento de Fibroblastos , Fibroblastos/citologia , CamundongosRESUMO
BACKGROUND: Werner protein (WRN) has DNA helicase activity and participates in recombination, replication and repair of DNA. Loss-of-function mutations in WRN gives rise to genetic instability and diseases such as premature ageing and cancer. Upregulation of WRN promotes proliferation and survival of cancer cells. AIM: To evaluate the expression pattern of WRN in closely related skin cancers and their correlation with age, sex and UV exposure. METHODS: Immunohistochemistry was used to investigate expression of WRN in formalin-fixed, paraffin wax-embedded tissue specimens of 9 squamous cell carcinoma (SCC), 15 actinic keratosis (AK), 11 Bowen disease (BD) and 11 normal-appearing peripheral tissue samples, obtained from patients during surgical resections. RESULTS: WRN expression was significantly increased in BD, AK and SCC compared with normal controls, with the mean WRN staining score being highest in BD, followed by AK and SCC. However, age, sex and sun exposure were not associated with WRN expression. CONCLUSIONS: To our knowledge, this is the first report to date investigating the expression of WRN in skin cancers. The overtly high expression of WRN in premalignant lesions and in in situ cancer, with relatively low WRN expression in SCC, may indicate that WRN contributes as a checkpoint for early DNA damage response in skin tumorigenesis.
Assuntos
Doença de Bowen/metabolismo , Carcinoma de Células Escamosas/metabolismo , Dano ao DNA , Exodesoxirribonucleases/metabolismo , Ceratose Actínica/metabolismo , RecQ Helicases/metabolismo , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Doença de Bowen/genética , Doença de Bowen/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Ceratose Actínica/genética , Ceratose Actínica/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Helicase da Síndrome de WernerRESUMO
Cell culture of human-derived neural stem cells (NSCs) is a useful tool that contributes to our understanding of human brain development and allows for the development of therapies for intractable human brain disorders. Human NSC (hNSC) cultures, however, are not commonly used, mainly because of difficulty with consistently maintaining the cells in a healthy state. In this study, we show that hNSC cultures, unlike NSCs of rodent origins, are extremely sensitive to insulin, an indispensable culture supplement, and that the previously reported difficulty in culturing hNSCs is likely because of a lack of understanding of this relationship. Like other neural cell cultures, insulin is required for hNSC growth, as withdrawal of insulin supplementation results in massive cell death and delayed cell growth. However, severe apoptotic cell death was also detected in insulin concentrations optimized to rodent NSC cultures. Thus, healthy hNSC cultures were only produced in a narrow range of relatively low insulin concentrations. Insulin-mediated cell death manifested not only in all human NSCs tested, regardless of origin, but also in differentiated human neurons. The underlying cell death mechanism at high insulin concentrations was similar to insulin resistance, where cells became less responsive to insulin, resulting in a reduction in the activation of the PI3K/Akt pathway critical to cell survival signaling.
Assuntos
Técnicas de Cultura de Células , Insulina/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Apoptose , Diferenciação Celular , Meios de Cultura , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neurais/citologia , Transdução de SinaisRESUMO
The pointing instability of energetic electron beams generated from a laser-driven accelerator can cause a serious error in measuring the electron spectrum with a magnetic spectrometer. In order to determine a correct electron spectrum, the pointing angle of an electron beam incident on the spectrometer should be exactly defined. Here, we present a method for absolutely calibrating the electron spectrum by monitoring the pointing angle using a scintillating screen installed in front of a permanent dipole magnet. The ambiguous electron energy due to the pointing instability is corrected by the numerical and analytical calculations based on the relativistic equation of electron motion. It is also possible to estimate the energy spread of the electron beam and determine the energy resolution of the spectrometer using the beam divergence angle that is simultaneously measured on the screen. The calibration method with direct measurement of the spatial profile of an incident electron beam has a simple experimental layout and presents the full range of spatial and spectral information of the electron beams with energies of multi-hundred MeV level, despite the limited energy resolution of the simple electron spectrometer.
RESUMO
Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53.
Assuntos
Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas de Fluorescência Verde/metabolismo , Humanos , Leupeptinas/farmacologia , Mitocôndrias/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ligação Proteica , Estabilidade Proteica , Proteólise , Domínios RING Finger , Proteínas Recombinantes de Fusão/metabolismo , Proteína Supressora de Tumor p53/fisiologia , UbiquitinaçãoRESUMO
Simultaneous liver and kidney transplantation (SLKT) is now considered the treatment of choice for patients with concurrent end-stage liver and kidney diseases. Even though the early postoperative mortality rate following SLKT is reported to be high compared to that of liver transplantation alone, the liver graft from the same donor has been argued to induce better kidney graft acceptance as evidenced by a low rate of acute renal rejection episodes. There have been many reports of a low incidence of acute renal rejection following SLKT; however, only a few cases were proven by simultaneous biopsies. The authors experienced a case of biopsy-proven isolated acute cellular rejection of the liver graft following SLKT.
Assuntos
Rejeição de Enxerto/diagnóstico , Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Transplante de Fígado/imunologia , Hemissuccinato de Metilprednisolona/uso terapêutico , Doença Aguda , Biópsia , Humanos , Inflamação , Transplante de Rim/patologia , Circulação Hepática , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoAssuntos
Corioamnionite/diagnóstico , Conteúdo Gastrointestinal/química , Doenças do Prematuro/diagnóstico , Interleucina-6/análise , Adulto , Biomarcadores/análise , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Projetos Piloto , Gravidez , Estudos Prospectivos , Cordão UmbilicalRESUMO
Vitreoscilla hemoglobin (VHb) has been successfully used to enhance production of foreign proteins in several microorganisms including Escherichia coli. We compared the expression of an oxygen-dependent foreign protein, green fluorescent protein (GFP) under co-expression of VHb in two typical industrial E. coli strains, BL21 (a B derivative) and W3110 (a K12 derivative), which have different metabolic properties. We employed the nar oxygen-dependent promoter for self-tuning regulation of VHb expression due to the natural transition of dissolved oxygen (DO) level during culture. We observed several interesting and differing behaviors in cultures of the two strains. VHb co-expression showed a positive influence on expression, and even on solubility, of GFP in both strains; while strain BL21 had the higher GFP expression level, W3110 showed higher solubility of expressed GFP. GFP expression in strain BL21 was very largely affected by variation of aeration environments, but W3110 was not significantly impacted. We surmised that this arose from different oxygen utilization abilities and indeed the two strains showed different patterns of oxygen uptake rate. Interestingly, the VHb co-expressing W3110 strain exhibited a peculiar increasing pattern of GFP expression during the late culture period even under low aeration conditions and this enhancement was more obvious in large-scale cultures. Therefore, this strain could be successfully employed in practical large-scale production cultures where DO levels tend to be limited.
Assuntos
Escherichia coli/metabolismo , Hemoglobinas/metabolismo , Proteínas Luminescentes/metabolismo , Vitreoscilla/metabolismo , Meios de Cultura , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde , Hemoglobinas/genética , Microbiologia Industrial/métodos , Proteínas Luminescentes/genética , Consumo de Oxigênio , Regiões Promotoras Genéticas , Transcrição Gênica , Vitreoscilla/genéticaAssuntos
Baculoviridae/genética , Vetores Genéticos , Biologia Molecular/métodos , Proteínas Virais/genética , Animais , Regulação Viral da Expressão Gênica , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Vírus de Insetos/genética , Insetos , Larva , Proteínas Luminescentes/genética , Proteínas de Matriz de Corpos de Inclusão , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Estruturais ViraisRESUMO
Fas/Fas ligand system triggers apoptosis in many cell types. Bcl-XL overexpresion antagonizes Fas/Fas ligand-mediated cell death. The mechanism by which Bcl-XL influences Fas-mediated cell death is unclear. We have found that microtubule-damaging drugs (e.g. Paclitaxel) induce apoptosis in a Fas/FasL-dependent manner. Inhibition of Fas/FasL pathway by anti-FasL antibody, mutant Fas or a dominant negative FADD blocks paclitaxel-induced apoptosis. Paclitaxel induced apoptosis through activation of both caspase-8 and caspase-3. Overexpression of Bcl-XL leads to inhibition of paclitaxel-induced FasL expression and apoptosis. Bcl-XL prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes) by inhibiting the activation of calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT for it to move to the nucleus. The loop domain in Bcl-XL can suppress the anti-apoptotic function of Bcl-XL and may be a target for regulatory post-translational modifications. Upon phosphorylation, Bcl-XL loses its ability to bind with calcineurin. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, paclitaxel and other drugs that disturb microtubule function kill cells, at least in part, through the induction of FasL, and Bcl-XL-mediated resistance to these agents is related to failure to induce FasL expression.
Assuntos
Apoptose , Glicoproteínas de Membrana/biossíntese , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Calcineurina/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Proteína Ligante Fas , Humanos , Células Jurkat , Ligantes , Glicoproteínas de Membrana/genética , Fatores de Transcrição NFATC , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Proteína bcl-X , Receptor fas/metabolismoRESUMO
Plasmids containing an antisense fragment of the sigma(32) gene were constructed and introduced into Escherichia coli cells. Downregulation of the sigma(32)-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH). Northern blot analyses revealed that sigma(32) sense mRNA was virtually undetected in antisense-producing cultures from 5 to 20 min after antisense induction. However, lower-molecular-weight bands were found, presumably due to partial degradation of sigma(32) mRNA. While a >10-fold increase in sigma(32) protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of sigma(32). Correspondingly, antisense synthesis resulted in a decreased level of a sigma(32) regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without sigma(32) antisense mRNA. The total yield of OPH in the presence of sigma(32) antisense was, on average, 62% of the yield without antisense. However, during sigma(32) antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures.
Assuntos
Escherichia coli/metabolismo , Esterases/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fator sigma/genética , Fatores de Transcrição/genética , Arildialquilfosfatase , Sequência de Bases , Escherichia coli/enzimologia , Escherichia coli/genética , Esterases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Cinética , Plasmídeos , RNA Mensageiro/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5' end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.
Assuntos
Esterases/metabolismo , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Arildialquilfosfatase , Sequência de Bases , Cromatografia de Afinidade/métodos , Primers do DNA , Escherichia coli/genética , Esterases/genética , Esterases/isolamento & purificação , Proteínas de Fluorescência Verde , Espectrometria de FluorescênciaRESUMO
To understand the differences in production and secretion patterns between plasmid-harboring and chromosome-integrated recombinant yeasts, the two recombinant Saccharomyces cerevisiae yeasts, containing the structural glucoamylase STA gene and the SUC2 promoter, were investigated. Both systems were regulated by glucose concentration in the culture broth. First, the glucoamylase activity per gene copy number of the chromosome-integrated recombinant yeast was 2.8- to 5.6-fold higher than that of the plasmid-harboring recombinant yeast. Overburdened owing to high copy number, the plasmid-harboring recombinant yeast gave lower glucoamylase activity per gene copy number. Second, the efficiency of signal sequence was compared; the secretion efficiency of glucoamylase in the plasmid-harboring recombinant yeast was higher than that in the chromosome-integrated recombinant yeast at 96 h of cultivation (74 vs 65%). We postulated that the higher level of secretion efficiency of the plasmid-harboring recombinant yeast resulted because the production level did not reach the capacity of the secretory apparatus of the host yeast. However, the specific secretion rate was much higher in the chromosome-integrated recombinant yeast even though the final secretion efficiency was lower. The lower secretion rate in the plasmid-harboring recombinant yeast could be explained by an adverse effect caused by higher production rate. Finally, the optimal glucose concentration for glucoamylase production in the chromosome-integrated recombinant yeast culture was lower than that in the plasmid-harboring recombinant yeast culture owing to gene dosage effect.
Assuntos
Cromossomos Fúngicos , Glucana 1,4-alfa-Glucosidase/biossíntese , Glucana 1,4-alfa-Glucosidase/metabolismo , Glicosídeo Hidrolases/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Divisão Celular , Fracionamento Celular , Clonagem Molecular , Fermentação , Plasmídeos/metabolismo , Recombinação Genética , Espectrofotometria , Fatores de Tempo , beta-FrutofuranosidaseRESUMO
A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-cell-density cultivation of Escherichia coli. A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E. coli JM105 was developed. A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity. The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-beta-D-thiogalactopyranoside (IPTG) induction system. Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay). Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized.
Assuntos
Escherichia coli/citologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Algoritmos , Arabinose/farmacologia , Biomassa , Divisão Celular/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Simulação por Computador , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Retroalimentação , Corantes Fluorescentes/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Glucose/metabolismo , Proteínas de Fluorescência Verde , Isopropiltiogalactosídeo/farmacologia , Cinética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Modelos Biológicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.
Assuntos
Escherichia coli/genética , Engenharia Genética , Proteínas Luminescentes/genética , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Insulin-like growth factor II (IGF-II), highly expressed in a number of human tumours, has been recently known to promote neovascularization in vivo. Yet, the detailed mechanism by which IGF-II induces angiogenesis has not been well defined. In the present study, we explored an angiogenic activity of IGF-II in in vitro angiogenesis model. Human umbilical vein endothelial cells (HUVECs) treated with IGF-II rapidly aligned and formed a capillary-like network on Matrigel. In chemotaxis assay, IGF-II remarkably increased migration of HUVECs. A rapid and transient activation of p38 mitogen-activated protein kinase (p38 MAPK) and p125 focal adhesion kinase (p125FAK) phosphorylation was detected in HUVECs exposed to IGF-II. IGF-II also stimulated invasion of HUVECs through a polycarbonate filter coated with Matrigel. Quantitative gelatin-based zymography identified that matrix metalloproteinase-2 (MMP-2) activity generated from HUVECs was increased by IGF-II. This induction of MMP-2 activity was correlated with Northern blot analysis, showing in HUVECs that IGF-II increased the expression of MMP-2 mRNA, while it did not affect that of TIMP-2, a tissue inhibitor of MMP-2. These results provide the evidence that IGF-II directly induces angiogenesis by stimulating migration and morphological differentiation of endothelial cells, and suggest that IGF-II may play a crucial role in the progression of tumorigenesis by promoting the deleterious neovascularization.
Assuntos
Movimento Celular/fisiologia , Quimiotaxia , Fator de Crescimento Insulin-Like II/farmacologia , Neovascularização Patológica/fisiopatologia , Transformação Celular Neoplásica , Células Cultivadas , Células Epiteliais , Humanos , Invasividade Neoplásica/fisiopatologia , Cordão Umbilical/irrigação sanguínea , Cordão Umbilical/citologiaRESUMO
Membrane-type 1 matrix metalloproteinase (MT1-MMP) expressed in tumor cells is believed to be important for the pericellular degradation of extracellular matrices during invasion and metastasis. To analyze the mechanism by which MT1-MMP becomes expressed in cancer cells, we assessed the MT1-MMP promoter region for the presence of cis-acting promoter elements that support transcription in transformed cells. Our tumor model consisted of Madin-Darby canine kidney (MDCK) cells transformed by v-src (src4 cells). MT1-MMP mRNA was only faintly detected in parental cells but was strongly expressed in the src4 cells. In parallel, src4 cells invaded into collagen gels, whereas MDCK cells did not. When MDCK and src4 cells were transiently transfected with a plasmid containing of -3000 to -99 nt from the upstream region of the MT1-MMP gene, the promoter activity was 2.6-fold higher in src4 cells than in MDCK cells. Furthermore, the region between -399 and -356 nt was found to contain the src4-specific enhancer element(s). Tandem Sp1 binding sites were also found to be essential in promoting transcription. An Egr-1 site that partially overlaps with the Sp1 sites was found to cooperate with the src4-specific enhancer and to also contribute weakly to the basal promoter activity. The presence of transcription factors that bind to the src4-specific enhancer site was detected by mobility-shift assays in src4 cell nuclear extracts but only weakly in MDCK extracts. Thus, we have identified a novel enhancer element that acts specifically in the transformed cells to enhance MT1-MMP expression.